PROJECT SUMMARY The innate immune system of mosquitoes is a critical determinant of their vector competence. This includes the ability to support development and transmission of the protozoan parasite species in the genus Plasmodium by Anopheles mosquitoes, the principal vectors of human malaria world-wide. Insight into the regulation of innate immune effector mechanisms remains incomplete, but is vitally important to our fundamental understanding of host-pathogen interactions in this most important human vector-borne disease. The long-term goal is to understand immune system regulation in An. gambiae to inform current and future vector control strategies. The objective of this application is to globally identify mechanisms of immune system regulation by determining the interactions within the extracellular protease network that activate and link opsonization to melanization in the context of distinct microbial infections. The rationale for the proposed research is that detailed information on the protease network that regulate mosquito immunity could be employed to predict long-term efficacy of novel vector control strategies that employ microbial agents, and manipulate infection outcome. Guided by our preliminary data, the following three specific aims will be pursued: (1) Determine the interactions of proteases and their homologs that are critical for mosquito immunity; (2) Assess the impact of the protease network on immunity and mosquito fitness; and (3) Visualize the immunoregulatory network in mosquitoes using network science. Under the first aim, we will test the hypothesis that clip-serine proteinases and their homologs form functional modules that are required for optimal immune responses by defining their cleavage patterns, genetic interactions, and precise biochemi-cal function. Under the second aim, the potential effect of the protease network on pathogen resistance and tolerance as well as mosquito fitness will be assessed using common microbial challenge models and life table analyses. Under the third aim standard network science approaches will be used to visualize all protease interactions in the system as a static multilayered network and to analyze this network to infer proteolytic flow through that links opsonization and melanization and to identify the key molecules that control immunity. The proposed research is innovative, as it will for the first time evaluate protease cascades as a single, integrated network that controls mosquito humoral immunity during diverse immune challenges. Additionally, this project will use network science as a highly innovative approach to the study of mosquito innate immunity, which if successful will be transformative to the field of insect immunology. This project is significant as it will provide comprehensive understanding of the contribution of the protease network to mosquito health as well as the limitations of the system in overcoming infection. Ultimately, this knowledge could be employed to manipulate infection outcome and thus inform the development of new disease control strategies that aim at disrupting malaria parasite development in its vector.